Methane production by Methanothrix thermoacetophila via direct interspecies electron transfer with Geobacter metallireducens

ABSTRACT Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila. Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c-type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila. Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. IMPORTANCE Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.


Supplementary Text Carbon metabolism of Mx. thermoacetophila grown under various conditions
Many carbon metabolism genes were highly expressed by Mx. thermoacetophila cells grown under both DIET and acetate conditions.In all of the conditions, most genes from pathways for methanogenesis from acetate and CO2, carbon fixation, the reductive citric acid cycle, and carbon monoxide and formate metabolism had RPKM values that were more than 2-fold above the median RPKM values (Figure 6 and Table S3).Addition of magnetite also significantly increased the expression of almost all of these genes (Figure 6 and Table S3).

Acetoclastic pathway
Genes coding for proteins from the acetoclastic pathway, acetyl-CoA synthetase (acs) and CO dehydrogenase/acetyl-CoA synthase (cdh), were highly expressed in all 4 conditions (Figure 6 and Table S3).There are 4 putative acetyl-CoA synthetase (acsA) genes in the Mx.thermoacetophila genome.Similar to previous reports (1), acsA-1 (Mthe_1194) was the most highly expressed and had RPKM values that were 105 to 255 (p-values < 9.0×10 -6 ) times higher than the median.Genes coding for the delta and gamma subunits from the acetyl-CoA decarbonylase/synthase complex (cdhDE; Mthe_0287-0288) were also 39 to 133 (p-values < 0.002) times more highly expressed than the median RPKM values.Addition of magnetite significantly increased expression of these genes (Figure 6 and Table S3).

Carbon fixation
Methanothrix species have a RuBisCO-mediated carbon fixation pathway (reductive hexulose phosphate (RHP) pathway) that forms formaldehyde as an intermediate (2).The Mx. thermoacetophila genome also has two genes (Mthe_0988 and Mthe_1603) that code for formaldehyde-activating enzyme (Fae), an enzyme that catalyzes the conversion of formaldehyde into 5,10-methylenetetrahydromethanopterin, an intermediate in the methanogenic CO2 reduction pathway.In addition to duplicate copies of fae, the Mx.thermoacetophila genome has multiple copies of many of the genes from the RHP pathway, several of which are fusion proteins.One of the fae genes is fused to another gene coding for 3-hexulose-6-phosphate synthase (hps), which is the enzyme in the RHP pathway that catalyzes formation of formaldehyde and D-ribulose-5-phosphate from D-arabino-3hexulose-6-phosphate.Other Methanothrix species also have fae/hps fusion genes, and it has been proposed that this fusion minimizes carbon loss because formaldehyde released from the RHP pathway can be directly fed into the methanogenesis pathway (2).
Similar to studies of Methanothrix species participating in DIET in GAC-amended reactors (3), Mx. thermoacetophila was highly expressing most of the RHP pathway genes in all conditions (Figure 6 and Table S3).Addition of magnetite significantly enhanced expression of many of these genes.In particular, the gene coding for ribulose-bisphosphate carboxylase (RuBisCO, Mthe_1616), which is the enzyme responsible for the first step in the carbon fixation pathway was 2.7 (p-value = 1.04×10 -6 ) and 5.6 (p-value = 5.38×10 -13 ) times more highly expressed in the presence of magnetite when cells were grown by DIET or acetoclastic methanogenesis, respectively (Supplementary Table S4).
In addition to the RHP pathway, the genome of Mx. thermoacetophila has a gene coding for ATP-citrate lyase (acly; Mthe_1476), which can form acetyl-CoA and oxaloacetate from citrate and plays a major role in the reductive TCA cycle (4)(5)(6).This gene was 4.6 to 8.1 times higher (p-value < 0.003) than the median RPKM values in all of the conditions (Figure 6 and Table S3).High expression of acly suggests that acetyl-CoA was present in cells even during growth by DIET, which helps to explain why acetoclastic genes were being expressed at high levels by DIET-grown cells.

Carbon dioxide reduction pathway
Mx. thermoacetophila is not capable of hydrogenotrophic growth (7) as it does not have any hydrogenase proteins (8) and hydrogenase activity has not been detected in Mx. thermoacetophila membranes (9).However, it does have genes that code for proteins from the CO2 reduction pathway, which is the pathway used by hydrogenotrophic methanogens for methane production (8).Previous studies have shown that these genes are highly expressed by DIET-grown Methanothrix cells (10,11).However, transcriptomic comparisons of CO2 reduction genes between DIET-grown and acetoclastic cells were not done in these previous experiments.
The elevated expression of CO2 reduction pathway genes may be explained by the finding that genes involved in the formation of formate and carbon monoxide were also highly expressed and both of these intermediates feed into the CO2 reduction pathway (Figure 6).Mx. thermoacetophila cannot utilize CO or formate as substrates for methanogenesis (7,12), however, genes coding for formate dehydrogenase and carbon monoxide dehydrogenase are found in the genome.Genes from an operon with two genes coding for the alpha subunit of formate dehydrogenase (fdhA; Mthe_0915, Mthe_0913) and an FdhB-like ferredoxin reductase protein (Mthe_0914) with an FAD-binding site that could bind coenzyme F420 were highly expressed in all of the conditions; 4 to 7 times higher than median RPKM values (p-values < 0.007).The enzyme complex encoded by this gene cluster could potentially oxidize formate to CO2 and transfer electrons to F420 to provide a source of reduced F420 for methanogenesis.